Human Induced Pluripotent Stem Cell-Derived 3D-Neurospheres are Suitable for Neurotoxicity Screening

Abstract

We present a hiPSC-based 3D in vitro system suitable to test neurotoxicity (NT). Human iPSCs-derived 3D neurospheres grown in 96-well plate format were characterized timewise for 6-weeks. Changes in complexity and homogeneity were followed by immunocytochemistry and transmission electron microscopy. Transcriptional activity of major developmental, structural, and cell-type-specific markers was investigated at weekly intervals to present the differentiation of neurons, astrocytes, and oligodendrocytes. Neurospheres were exposed to different well-known toxicants with or without neurotoxic effect (e.g., paraquat, acrylamide, or ibuprofen) and examined at various stages of the differentiation with an ATP-based cell viability assay optimized for 3D-tissues. Concentration responses were investigated after acute (72 h) exposure. Moreover, the compound-specific effect of rotenone was investigated by a panel of ER-stress assay, TUNEL assay, immunocytochemistry, electron microscopy, and in 3D-spheroid based neurite outgrowth assay. The acute exposure to different classes of toxicants revealed distinct susceptibility profiles in a differentiation stage-dependent manner, indicating that hiPSC-based 3D in vitro neurosphere models could be used effectively to evaluate NT, and can be developed further to detect developmental neurotoxicity (DNT) and thus replace or complement the use of animal models in various basic research and pharmaceutical applications.

Keywords: 3D culture; induced pluripotent stem cells; neurite outgrowth; neurospheres; neurotoxicity.

Figure 1

Growth properties of 3D neurospheres during the 6 weeks of differentiation (A) Representative microscopic view of 3D spheroids, investigated at weekly intervals (4×, scale bar: 200 µm). (B) The average diameter of 3D spheroids in µm measured by CellSens Dimension software (Olympus) (n = 3, in each experiment 96 spheroids were compared weekly trough 6-weeks). (C) Average total protein content of spheroids determined by Pierce BCA Protein Assay. Note that D2, D7 and D14 samples were measured by pooling three spheroids and individual values were calculated, while in all other timepoints spheroids were measured individually (n = 3, in each stage 24 spheroids were measured). ±SEM values are presented on graphs. (* p < 0.05).

Figure 2

Immunocytochemical analysis of 3D spheroids. (A) Spheroids were fixed and cryosectioned then immunostained at weekly intervals from D2 until D42 stage. The first line represents the overview of the cryosectioned spheroids, while the rest of the panel shows higher magnifications. Relevant markers of proliferation (KI67), neural stem cells (NESTIN), neuronal differentiation (TUBB3 and MAP2), an intermediate filament of dendrites and axons (NF200), synaptic vesicles of neurons (VAMP2), astrocyte (AQP4), and oligodendrocyte (MBP) specific proteins were stained. Protein name IDs are indicated with colors, representing the color of the fluorophore used (e.g., green as Alexa 488; red as Alexa 594) Nuclei were counterstained with DAPI (in blue). Scale bar: 100 µm (first line only) and 25 µm. (B) Quantitative analysis of the immunostainings on confocal images. The numbers of Ki-67, NESTIN, AQP4, TUBB3, NF200kD, MBP, VAMP2, and MAP2 immunoreactive pixels were measured in 5 neurospheres (middle sections, 5 randomly selected fields/slide) at every time points. Data was normalized with DAPI positive nuclei number. Data were expressed as percentage of marker/DAPI ratio ± SEM (* p < 0.05).

Figure 3

Real-time PCR measurements of relevant markers during the neuronal differentiation of the spheroids. Twelve 3D spheroids were pooled, lysed with RLT-buffer and used in RT-PCR analysis at each timepoints. Where the genes are already expressed in D2 samples, it is referred as 1. Day 2, 7, 14 and 21 values for GRIN1, CHAT, TH, GAD1, and SLC6A4 are not indicated as these genes were not expressed at those time points. Graphs represent normalized relative expression values, analyzed for each gene using the ddCT method [41]. Mean values and ± SEM of three biological replicates (n = 3) are presented on graphs, significance are determined by paired T test (∗ p < 0.01). Note that y axis’s scales alter according to the different relative expression values.

Figure 4

Immunocytochemical detection of neuronal subtypes in 3D neurospheres. Presence of synapses was determined with post-synaptic marker PSD95 and synaptic protein Synaptophysin (SYNP) double staining. Glutamatergic (VGLUT1/2), GABAergic (GAD65/67), cholinergic (VAChT) and dopaminergic (TH) neurons were detected in the developing 3D neurospheres from D28. All samples were stained with TUBB3 (in red) to label the neurites. Protein name IDs are indicated with colors, representing the color of the used fluorophore (e.g., green as Alexa 488; red as Alexa 594). Nuclei were counterstained with DAPI (in blue). Scale bar: 25 µm.

Figure 5

Cell viability measurement on D21 3D neurospheres after 72 h exposure. (A) Exposure scheme at D21 stage. (B) Concentration response curves of compounds, tested in 7 different concentrations (see concentrations listed in Table S3), representing the cell viability (%) of treated D21 3D neurospheres (n = 3). Concentration values are presented in log µM ± SEM. EC10 and EC50 values are presented on graphs where applicable.

Figure 6

Cell viability measurement on D28 3D neurospheres after 72 h exposure with toxicants. (A) exposure scheme at D28 stage. (B) Concentration-response curves of compounds, tested in 7 different concentrations (see concentrations listed in Table S3), representing the cell viability (%) of treated D28 3D neurospheres (n = 3). Concentration values are presented in log µM ± SEM. EC10 and EC50 values are presented on graphs where applicable.

Figure 7

Cell viability measurement on D42 3D neurospheres after 72 h exposure with toxicants. (A) exposure scheme at D42 stage. (B) Concentration-response curves of compounds, tested in 7 different concentrations (see concentrations listed in Table S3), representing the cell viability (%) of treated D42 3D neurospheres (n = 3). Concentration values are presented in log µM ± SEM. EC10 and EC50 values are presented on graphs where applicable.

Figure 8

Comparison of the toxic effect of compounds. (A) Ranking of compounds at different differentiation stages based on EC50 values. Darker background color represents higher toxicity, while white color represents non-toxic compounds. (B) Radar chart showing the comparison of compounds based on the order of EC10 or EC50 values, at various differentiation stages (yellow line with rectangular: D21; orange line with circle: D28; green line with square: D42) (see also EC values in Table S4). Concentration values are presented in log µM.

Figure 9

Effect of Rotenone (ROT) exposure on 3D neurospheres at three differentiation stages. (A) 3D neurospheres were treated with 0.5 µM ROT concentration for 72 h at three different maturation timepoints (D21, D28, and D42), fixed, sectioned and analyzed to detect the cellular effect of ROT by TUNEL assay (DeadEnd™ Colorimetric TUNEL System, Promega), compared to the vehicle (0.1% DMSO) treated control (scale bar: 100 µm). (B) ROT treatment revealed in average a 15% increase in the apoptotic cell number compared to the control in each stage (* p < 0.05). Average values are presented on graphs (n = 3). (C) Ultrastructure of mitochondria in control (panel a, b, c) and ROT treated (d–i) neurons in the 3D spheroids. See the alteration of the internal membrane (cristae) morphology (white arrowheads) in ROT treated cells (d, e, h, i). Black star: unidentifiable cristae morphology in lighter mitochondria (h, i); black arrowhead: membrane swirl in darker organelles (e, f); white arrows: matrix with and without matrix granules (control cells: a, c; treated cells: g, i) Note the density difference between these granules in control (c) and ROT treated mitochondria (panel i) (D21: a, d, f, h; D28: i; D42: b, c, g) (scale bar: 250 nm).

Figure 10

Neurite outgrowth measurement on D21 3D spheroids, exposed for 72 h with ROT. (A) Representative photograph of control (untreated) and ROT (0.5 µM) treated D21 spheroid immunolabeled with TUBB3 (in green). White lines represent the border of the spheroids where the neurite outgrowth was determined from, using ImageJ software (scale bar: 100 µm). (B) Total neurite length/spheroid (presented in µm ± SEM) and the average number of neurites/spheroids were determined 24 h after plating the treated spheroids. Different symbols denote treatment groups. (n = 3, in each experiment 8 spheroids were treated in each group) (** p < 0.01).